Angiogenesis is a rate-limiting step in the progression of human tumors. Key proteins involved in tumor vessel formation are good targets for therapy, since the forming vasculature is composed of genetically stable cells unlike the malignant cells in the tumor proper. The vascular endothelial growth factor (VEGF) receptors are thought to be master regulators of embryonic and tumor angiogenesis; thus, are currently targets for anti-angiogenic therapy. The transparency of the zebrafish embryo makes it an ideal model for angiogenic studies since blood flow can be easily observed in a living animal. In preliminary studies, a human VEGF receptor inhibitor, PTK787/ZK222584, was used to induce potent inhibition of embryonic vessels in the zebrafish. This chemcial genetic approach allowed an examination of the VEGF receptor signaling pathway as an upregulation of a downstream effector, AKT/PKB, can override the receptor block to provide a robust vessel rescue (Chan et al., 2002). In this grant proposal, chemical inhibition is combined with proven forward genetics to identify critical genes in angiogenic signaling as enhancers or suppressors of the anti-angiogenic phenotype. A pilot screen has been conducted using F2 heterozygous embryos under the influence of PTK787. Angiogenic defects have been confirmed genetically in F3 homozygous recessive embryos. Thus, a large scale screen will allow us to identify physiologically relevant players in the VEGF signaling pathway as additional targets for inhibitor therapy in cancer treatments. The application has two specific aims. Aim 1. To perform an enhancer screen for angiogenic mutations under drug-sensitized VEGF receptor function. Aim 2. To perform a suppressor screen for mutations that can override the drug-induced anti-angiogenics effects in zebrafish embryos.